Project 3 Summary Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma characterized by aberrant genetic and epigenetic events. A variety of therapeutic strategies have been used to improve the outcome of MCL patients including immune-chemotherapy and stem cell transplantation. Approaches targeting the B cell receptor (BCR) signaling pathway with ibrutinib have shown promise, however, outcome data is immature and emerging resistance is clearly evident. Thus there remains a clear, unmet need for novel therapeutic approaches for treating MCL. We have shown that PRMT5 is selectively over expressed in MCL and drives global repressive epigenetic marks on chromatin to silence regulatory and tumor suppressor genes. PRMT5 knock down with shRNA promotes selective MCL toxicity which led us to hypothesize that PRMT5 is an attractive target for this disease. We have developed a ?first-in-class? drug that selectively inhibits PRMT5 activity with IC50 in low nM range. Our lead drug is toxic to lymphoma, not normal B cells, is well tolerated in vivo with impressive anti-tumor activity in preclinical animal models of MCL. PRMT5 inhibitors have been used as chemical probes to dissect out critical pathways utilized by MCL tumor cells. Preliminary whole genome mapping with antibodies specific for PRMT5 epigenetic marks and whole transcriptome sequencing (RNA-Seq, in collaboration with WCM) in presence and absence of PRMT5 inhibitors has revealed a broad range of signaling, survival and growth pathways to be governed by this enzyme. Our preliminary work has found that PRMT5 directly targets regulatory genes such as PTPROt, TRIB3, and PIK3IP1 which negatively regulate BCR and PI3K/AKT pathways. Inactivation of constitutive AKT activity leads to dephosphorylation of FOXO1, promoting its epigenetic regulation of anti- cancer gene expression. We discovered that PRMT5 inhibition leads to direct de-repression of multiple micro- RNAs that may deplete CYCLIN D1 leading to activation of RB and restoration of the RB/E2F regulatory network. Consequently, PRMT5 inhibition leads to RB/E2F-mediated silencing of EZH2, SUZ12 and EED, components of the epigenetic repressor PRC2 complex, a family of lysine methyltransferase enzymes that are frequently mutated and activated in MCL. Thus, by inhibiting PRMT5, we can achieve direct inhibition of repressive arginine epigenetic marks while indirectly depleting lysine repressive marks like H3K27(Me3) and possibly, targets of FOXO1. Our strategy to inhibit a central player like PRMT5 has led to a novel approach to restore regulation to the MCL cell at multiple levels including the epigenome, BCR, PI3K/AKT, and CYCLIN D/RB-E2F pathways. We have proposed integrative whole genome approaches and strategies utilizing innovative preclinical MCL models to examine how PRMT5 inhibition can circumvent drug resistance encountered with novel molecularly targeted agents examined in clinical trials and preclinical work proposed in Projects 1 and 2. We present an innovative, straight-forward and robust approach to discover the genetic programs altered in MCL and will use this information to develop new strategies to treat this incurable disease.